The study of the effects of ethanol on protein phosphorylation in neurosecretory tissue will enhance our understanding of the deleterious impact of ethanol on biochemical events linking activation of membrane receptors to physiological responses such as hormone secretion. An endocrine cell line (AtT-20 cells) of anterior pituitary origin is used as a working model because it possesses multiple receptor types which regulate the synthesis and secretion of pro-opiomelanocortin (POMC) hormones (e.g., ACTH, alpha-MSH, beta-endorphin). The control of release and synthesis of POMC neuropeptides, as well as alterations in phosphorylated protein levels, is regulated in part by the adenylcyclase-cyclic adenosine monophosphate (AC/cyclic-AMP) system. Phosphorylated proteins obtained from broken-cell preparations or from intact cells are analyzed with single- and double- dimensional gel electrophoresis. Our findings indicate that phosphorylation can be stimulated in broken-cell preparations by cAMP, calmodulin, and phorbol esters. Furthermore, forskolin, which activates the AC/cycylic-AMP system and enhances POMC hormone synthesis and release, also induces an increase in the nuclear and cytoplasmic protein kinases in intact cells. Following additional characterization of the system, the effect of ethanol on protein phosphorylation will be examined.